Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas

Abstract

CRISPR-Cas is an efficient method for genome editing in organisms from bacteria to human cells. We describe a transgene-free method for CRISPR-Cas-mediated cleavage in nematodes, enabling RNA-homology-targeted deletions that cause loss of gene function; analysis of whole-genome sequencing indicates that the nuclease activity is highly specific.

Keywords: CRISPR; Caenorhabditis elegans; genome resequencing; reverse genetics; targeted nuclease.

Figure 1

A schematic of the CRISPR-Cas procedure. (A) An mRNA encoding the hCas9 nuclease fused to an SV40 DNA localization sequence is transcribed, capped, and polyadenylated in vitro. This message contains 5′- and 3′-UTRs demonstrated to function in the C. elegans germline (Wood et al. 2011). The 20-nucleotide sequence that will target the nuclease activity is cloned into the sgRNA vector, using Gibson recombinational cloning, and transcribed in vitro. (B) The mRNA and the sgRNA are injected into the syncytial gonads of adult hermaphrodites. The F₂ progeny of the injected hermaphrodites are examined to find animals showing the phenotype expected for homozygous loss-of-function of the targeted gene. (C) A fourth-larval-stage (L4) wild-type animal. (D) An L4 dpy-11(sy740) animal. Bar, 100 µm.